Fusion genes associated with acute megakaryoblastoc leukemias

ABSTRACT

The invention relates to human nucleotide sequences which occur as a result of the t(1;22)(p13;q13) chromosomal translocation event which is known to occur almost invariable in young children with acute megakaryoblastic leukemia. The translocation results in the formation of fusion genes which encode fusion proteins. The invention provides the nucleotide sequences of transcripts of the fusion genes and the amino acid sequences of the fusion proteins encoded thereby. Also provided are methods for detecting the t(1, 22) translocation, for identifying agents capable of binding to the fusion protein and for identifying agents useful for treating patients with acute megakaryoblastic leukemia.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with U.S. Government support underCancer Center Support (CORE) grants CA-21765 and CA-87064 from theNational Cancer Institute. The Government may have certain rights inthis invention.

FIELD OF THE INVENTION

The present invention is directed to the field of the molecular geneticsof cancer. Specifically, the present invention relates to human acutemegakaryoblastic leukemias in which a translocation between chromosomes1 and 22 (referred to in the art as “t(1;22)”) has occurred. On amolecular level, the DNA rearrangement in t(1;22) results in tworeciprocal fusion genes that each comprise segments of the RBM15 andMKL1 genes.

BACKGROUND OF THE INVENTION

Chromosomal abnormalities are frequently associated with malignantdiseases. In a number of instances, specific chromosomal translocationshave been characterized, which generate fusion genes encoding proteinswith oncogenic properties (Sawyers et al., Cell 64:337-350 (1991)).

Recently, the cloning of chromosomal translocations has led toidentification of pathogenically relevant oncogenic fusion transcriptsand proteins in specific subsets of acute nonlymphocytic leukemia(ANLL), such as promyelocytic leukemia-retinoic acid receptor alphafusion gene (PML-RARα) in acute promyelocytic leukemia (FAB-M3 subtype),the acute myeloid leukemia 1-eight twenty one fusion gene (AML1-ETO) inANLL with maturation (FAB-M2), and various mixed lineage leukemia (MLL)fusions in acute myelomonocytic and monocytic leukemias (FAB-M4 and -M5)(Melnick, A. & Licht, J. D., Blood 93, 3167-3215 (1999); Downing, J. R.,Br. J Haematol. 106, 296-308 (1999); Rowley, J. D., Semin. Hematol. 36,59-72 (1999); Look, A. T., Science 278, 1059-1064 (1997); Faretta, M.,Di Croce, L. & Pelicci, P. G., Sem. Hematol. 38, 42-53 (2001)). Despitethese significant advances, little is known about the genetic mechanismsunderlying acute leukemias of the megakaryoblastic (platelet precursor)lineage (AMKL, FAB-M7) (Cripe, L. D, infra). Almost invariably, AMKL innon-Down syndrome infants and young children harbor the t(1;22)(p13;q13)translocation, in most cases as the sole cytogenetic abnormality(Carroll, A. et al; Lion, T. et al., and Bernstein, J. et al., infra).Phenotypically, AMKL presents de novo (i.e., without a so-calledpreleukemic stage), with a large leukemia cell mass, and frequentfibrosis of bone marrow and other organs. Progression is usually rapiddespite therapy, with a median overall patient survival of only eightmonths. Thus, compositions and methods for the early and accuratediagnosis and treatment of leukemia, particularly AMKL, are needed.

SUMMARY OF THE INVENTION

Compositions and methods associated with the diagnosis and treatment ofleukemia are provided. The invention discloses the identification,cloning and sequencing of human nucleotide sequences corresponding tothe t(1;22)(p13;q13) chromosomal translocation event which occurs inindividuals with acute megakaryoblastic leukemia (AMKL). Therearrangement recombines sequences from the RNA-binding motifprotein-15gene (RBM15) on chromosome 1p13 with those from the MegakaryoblasticLeukemia-1 gene (MKL1) on chromosome 22q13. As a result of thet(1;22)(p13;q13) rearrangement, two fusion genes, one on each of the twoderivative chromosomes (der(1) and der(22)), are produced. The firstfusion gene, designated RBM15-MKL1, resides on der(22) and comprises a5′ portion of the RBM15 and a 3′ segment of the MKL1. The second fusiongene, designated as MKL1-RBM15, resides on der(1) and comprises a 5′portion of the MKL1 and a 3′ segment of the RBM15. Both fusion genes aretranscribed. A single transcript is expressed from RBM15-MKL1. Twotranscripts are found to be expressed from MKL1-RBM15. Isolatednucleotide molecules comprising the nucleotide sequences of theRBM15-MKL1 gene transcript and the two MKL1-RBM15 gene transcripts,MKL1-RBM15_(S) and MKL1-RBM15_(S+AE) are provided. Additionally providedare the amino acid sequences of the fusion proteins encoded by theRBM15-MKL1 transcript and the two MKL1-RBM15 transcripts.

Utilizing the sequences of the present invention, the present inventionprovides methods of identifying the presence of nucleic acids containingthe RBM15-MKL1 fusion gene and/or MKL1-RBM15 fusion gene and thetranscripts of these fusion genes in a sample. Such methods can be usedin diagnosis and treatment, for example, to determine if particularcells or tissues express RBM15-MKL1 or MKL1-RBM15 coding sequences, ordiagnostic assays to determine if a mammal has leukemia or a geneticpredisposition to (i.e., is at an increased risk of developing)leukemia.

The RBM15-MKL1 and MKL1-RBM15 fusion proteins and polypeptide sequencesof the invention, whether produced by host/vector systems or otherwise,can be used to produce antibodies which specifically recognize (i.e.,bind) the RBM15-MKL1 and MKL1-RBM15 fusion proteins, respectively. Theinvention provides methods of identifying the presence of nucleic acidsencoding the RBM15-MKL1 fusion protein and/or MKL1-RBM15 fusion proteinin a sample involving the use of such antibodies. Such methods find usein diagnosis and treatment of AMKL, for example, to determine ifparticular cells or tissues express the RBM15-MKL1 fusion protein and/orthe MKL1-RBM15 fusion proteins and to inhibit the activity of thesefusion proteins.

The present invention also provides for transgenic cells and animals,preferably mice, which (a) contain and express an RBM15-MKL1 fusion genederived from an exogenous source and subsequently introduced into thegenome of the cell or animal, (b) contain and express a gene encoding anRBM15-MKL1 fusion protein derived from an exogenous source andsubsequently introduced into the genome of the cell or animal, (c)contain and express an MKL1-RBM15 fusion gene derived from an exogenoussource and subsequently introduced into the genome of the cell or animal(d) contain and express a gene encoding an MKL1-RBM15 fusion proteinderived from an exogenous source and subsequently introduced into thegenome of the cell or animal, (e) contain and express both a geneencoding an MKL1-RBM15 fusion protein and a gene encoding an RBM15-MKL1fusion protein, with both genes derived from an exogenous source andsubsequently introduced into the genome of the animal, (f) knock-out theexpression of the RBM15 and/or MKL1 genes, or (g) knock-out theexpression of the RBM15-MKL1 and/or MKL1-RBM15 fusion proteins in anAMKL cell line. Methods of utilizing such cells and mice to identify andtest carcinogenic or therapeutic compositions are also described herein.

The nucleotide sequences of the RBM15-MKL1 and MKL1-RBM15 genes and thecoding sequences of the RBM15-MKL1 and the two MKL1-RBM15 fusionproteins of the invention can also be utilized to design and prepareagents which specifically inhibit the expression of the RBM15-MKL1 orMKL1-RBM15 genes in cells for therapeutic and other purposes. TheRBM15-MKL1 and MKL1-RBM15 nucleotide sequences of the invention can befurther utilized in methods of producing the RBM15-MKL1 and MKL1-RBM15fusion proteins, respectively.

The present invention further provides methods for isolating andidentifying the natural ligand(s) and gene targets bound by theRBM15-MKL1 and MKL1-RBM15 fusion proteins, and for identifyingderivatives of the ligand(s) or synthetic compounds that act to inhibitthe action of the RBM15-MKL1 and MKL1-RBM15 fusion proteins.

Additionally provided are compartmentalized kits to receive in closeconfinement one or more containers containing the reagents used in oneor more of the above described detection methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the normal RBM15 and MKL1proteins, and the fusion proteins formed by t(1;22). The locations ofthe fusion junctions in the proteins are indicated by vertical arrows.The 931-aa MKL1 protein (predicted mass, 98.9 kDa; SEQ ID. NO: 14)contains a bipartite nuclear localization signal (N, residues 14-31 ofSEQ ID NO: 14), a single SAP DNA-binding motif (residues 347-381 of SEQID NO: 14), a coiled-coil region (CC, residues 521-563 of SEQ ID NO:14), and a long C-terminal proline-rich segment (P, residues 564-811 ofSEQ ID NO: 14). The three isofomis of RBM15 (RBM15_(S), RBM15_(S+AE) andRBM15_(L)) share an identical 2863-bp 5′ coding sequence (nucleotides 84to 2946 of SEQ ID Nos. 7, 9, and 11) and differ only in their extremeC-termini, distal to the location of the t(1;22) fusion junction, due tosplicing of alternative exons. Specifically, RBM15_(L), the longestRBM15 transcript (approx. 9 kb), contains a unique 416-bp 3′ exon(nucleotides 2947 to 3362 of SEQ ID NO: 11) and encodes a predicted957-aa protein (mass, 104.6 kDa; SEQ ID NO: 12), whereas the shortesttranscript, RBM15_(S) (approx. 4 kb), possesses a divergent 3′ sequence(nucleotides 2947 to 3312 of SEQ ID NO: 7) and encodes a 969-aa protein(mass, 106.2 kDa; SEQ ID NO: 8). Detailed analysis of the approximately4-kb RBM15 transcript revealed a variant, RBM15_(S+AE) (short transcriptplus alternative exon), which contains an additional 111-bp exon(nucleotides 2947 to 3057 of SEQ ID NO: 9) interposed between the2,863-bp common sequence and the 366-bp sequence in RBM15_(S) andencodes a polypeptide of 977 aa (mass, 107.1 kDa; SEQ ID NO: 10). AllRBM15 isoforms contain three RNA recognition motifs (RRM; residues170-252, 374-451 and 455-529 of SEQ ID Nos. 8, 10, and 12), a bipartitenuclear localization signal (N, residues 716-733 of SEQ ID Nos. 8, 10,and 12), and a SPOC domain (S, residues 714-954 of SEQ ID Nos. 8, 10,and 12). Several regions in RBM15 of potential functional importance arecharacterized by a high content of specific aa, including aglycine/serine-rich segment (residues 60-166 of SEQ ID Nos. 8, 10, and12), a small proline-rich (LPPPPPPPLP) motif (residues 315-324 of SEQ IDNos. 8, 10, and 12), an arginine-rich portion (residues 616-732 of SEQID Nos. 8, 10, and 12), and a short C-terminal serine-rich segment(GSSDSRSSSSSAASD) at amino acids 865-879 of SEQ ID Nos. 8, 10, and 12.The 1883-aa RBM15-MKL1 chimeric protein (mass, 203.1 kDa; SEQ ID NO: 2)is comprised of the common N-terminal portion of RBM15 (residues 1 to954 of SEQ ID Nos. 8, 10, and 12) and all but the first two residues ofMKL1, thus containing all identified motifs of each normal protein. Bycontrast, the reciprocal MKL1-RBM15 fusions contain only the first twoaa of MKL1 fused to the short C-terminal sequences of either RBM15_(S)or RBM15_(S+AE), and are predicted to encode peptides of only 17 (SEQ IDNO: 4) and 25 (SEQ ID NO: 6) amino acids, respectively. The portions ofthe schematic illustrating the alternative C-termini of RBM15 and thepredicted MKL1-RBM15 fusion peptides are enlarged for clarity, and arethus not to scale.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods for the identification, diagnosis, andtreatment of leukemia or a genetic predisposition to leukemia areprovided. The present invention is based on the discovery of tworeciprocal fusion genes, RBM15-MKL1 and MKL1-RBM15, that result from thet(1;22)(p13;q13) chromosomal translocation event associated with acutemegakaryoblastic leukemia (AMKL). In particular, the invention providesthe novel nucleotide sequences of a transcript of the RBM15-MKL1 fusiongene (SEQ ID NO: 1) and two transcripts, MKL1-RBM15_(S) (SEQ ID NO: 3)and MKL1-RBM15_(S+AE) (SEQ ID NO: 5), of the MKL1-RBM15 fusion gene.Additionally provided are the amino acid sequences (SEQ ID NOS: 2, 4,and 6, respectively) of the fusion proteins encoded by such nucleotidesequences. Such nucleotide sequences and amino acid sequences find use,for example, in methods for detecting the t(1;22)(p13;q13) chromosomaltranslocation event associated with AMKL, methods for identifying agentsthat bind to the fusion proteins and methods for identifying agentsuseful for treating AMKL.

In addition to the RBM15-MKL1 and MKL1-RBM15 nucleotide and amino acidsequences, the invention provides isolated nucleotide moleculescomprising nucleotide sequences of three transcripts of the RBM15 gene,RBM15_(S) (SEQ ID NO: 7), RBM15_(S+AE) (SEQ ID NO: 9), and RBM15_(L)(SEQ ID NO: 11) and the nucleotide sequence of MKL1 (SEQ ID NO: 13).Further provided are isolated proteins comprising the amino acidssequences of the proteins encoded thereby, RBM15_(S) (SEQ ID NO: 8),RBM15_(S+AE) (SEQ ID NO: 10), and RBM15_(L) (SEQ ID NO: 12) and MKL1(SEQ ID NO: 14) respectively. Such nucleotide and amino acid sequencesfind use, for example, in methods for detecting the t(1;22)(p13;q13)chromosomal translocation event associated with AMKL and methods foridentifying agents useful for treating AMKL.

The RBM15 and MKL1 proteins, as well as the noted fusion proteinsderived from RBM15 and MKL1, are contemplated to act as transcriptionfactors which bind to corresponding DNA regulatory sequences. Thus theseproteins may be used to regulate the expression of genes that includethe regulatory DNA sequences that these proteins recognize. Methods foridentifying such regulatory DNA sequences based on their ability to bindRBM15, MKL1, RBM15-MKL1 and MKL1-RBM15 are also included in the presentinvention.

The nucleotide and amino acid sequences of the invention are set forthin the sequence listing. Below is a brief description of the sequencesin the sequences listing.

-   -   SEQ ID NO: 1 is the nucleotide sequence RBM15-MKL1 cDNA. The        open reading frame is from nucleotide 84 through nucleotide        5732.    -   SEQ ID NO: 2 is the amino acid sequence of the RBM15-MKL1 fusion        protein.    -   SEQ ID NO: 3 is the nucleotide sequence MKL1-RBM15_(S) cDNA. The        open reading frame is from nucleotide 551 through nucleotide        601.    -   SEQ ID NO: 4 is the amino acid sequence of the MKL1 -RBM15_(S)        fusion protein.    -   SEQ ID NO: 5 is the nucleotide sequence MKL1-RBM15_(S+AE) cDNA.        The open reading frame is from nucleotide 551 through nucleotide        625.    -   SEQ ID NO: 6 is the amino acid sequence of the MKL1-RBM15_(S+AE)        fusion protein.    -   SEQ ID NO: 7 is the nucleotide sequence RBM15_(S) cDNA. The open        reading frame is from nucleotide 84 through nucleotide 2990.    -   SEQ ID NO: 8 is the amino acid sequence of the RBM15_(S)        protein.    -   SEQ ID NO: 9 is the nucleotide sequence RBM15_(S+AE) cDNA. The        open reading frame is from nucleotide 84 through nucleotide        3014.    -   SEQ ID NO: 10 is the amino acid sequence of the RBM15_(S+AE)        protein.    -   SEQ ID NO: 11 is the nucleotide sequence RBM15_(L) cDNA. The        open reading frame is from nucleotide 84 through nucleotide        2954.    -   SEQ ID NO: 12 is the amino acid sequence of the RBM15_(L)        protein.    -   SEQ ID NO: 13 is the nucleotide sequence MKL1 cDNA. The open        reading frame is from nucleotide 551 through nucleotide 3346.    -   SEQ ID NO: 14 is the amino acid sequence of the MKL1 protein.    -   SEQ ID NO: 15 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated MKL1-294R.    -   SEQ ID NO: 16 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated MKL1-73R.    -   SEQ ID NO: 17 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated MKL1-59R.    -   SEQ ID NO: 18 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated RBM15(S)-2746F.    -   SEQ ID NO: 19 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated MKL1-204R.    -   SEQ ID NO: 20 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated MKL1-F.    -   SEQ ID NO: 21 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated RBM15(S)-2930R.    -   SEQ ID NO: 22 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated RBM15(L)-1636R.    -   SEQ ID NO: 23 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated RBM15-1118F.    -   SEQ ID NO: 24 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated RBM15-1551R.    -   SEQ ID NO: 25 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated RBM15-2831F.    -   SEQ ID NO: 26 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated RBM15-3149R.    -   SEQ ID NO: 27 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated RBM15-1616F.    -   SEQ ID NO: 28 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated RBM15-2004R.    -   SEQ ID NO: 29 is the nucleotide sequence of the synthetic        oligonucleotide primer herein designated MKL1-155F.

Based on these observations, one embodiment of the present inventionprovides a first isolated nucleotide molecule comprising the codingsequence (SEQ ID NO: 1) of the RBM15-MKL1 fusion protein, which encodesthe RBM15-MKL1 fusion protein (SEQ ID NO: 2), a second isolatednucleotide molecule, comprising the coding sequence (SEQ ID NO: 3) ofthe MKL1-RBM15_(S) fusion protein, which encodes the MKL1-RBM15_(S)fusion protein (SEQ ID NO: 4), a third isolated nucleotide moleculecomprising the coding sequence (SEQ ID NO: 5) of the MKL1-RBM15_(S+AE)fusion protein which encodes the MKL1-RBM15_(S+AE) fusion protein (SEQID NO: 6), a fourth isolated nucleotide molecule, comprising the codingsequence (SEQ ID NO: 7) of the RBM15_(S) protein, which encodes theRBM15_(S) protein (SEQ ID NO: 8), a fifth isolated nucleotide moleculecomprising the coding sequence (SEQ ID NO: 9) of the RBM15_(S+AE)protein which encodes the RBM15_(S+AE) protein (SEQ ID NO: 10), a sixthisolated nucleotide molecule, comprising the coding sequence (SEQ ID NO:11) of the RBM15_(L) protein, which encodes the RBM15_(L) protein (SEQID NO: 12), and a seventh isolated nucleotide molecule comprising thecoding sequence (SEQ ID NO: 13) of the MKL1 protein, which encodes theMKL1 protein (SEQ ID NO: 14).

It is recognized that nucleotide molecules and proteins of the inventionwill have a nucleotide or an amino acid sequence sufficiently identicalto a nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 or to anamino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14. The term“sufficiently identical” is used herein to refer to a first amino acidor nucleotide sequence that contains a sufficient or minimum number ofidentical or equivalent (e.g., with a similar side chain) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences have acommon structural domain and/or common functional activity. For example,amino acid or nucleotide sequences that contain a common structuraldomain having at least about 45%, 55%, or 65% identity, preferably 75%identity, more preferably 85%, 95%, or 98% identity are defined hereinas sufficiently identical.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, nonlimitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc.Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol.Biol. 215:403. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul el al.(1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0), which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.

The invention encompasses RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L), and MKL1 nucleic acid molecules andfragments thereof. Nucleic acid molecules that are fragments of thesenucleotide sequences are also encompassed by the present invention. By“fragment” is intended a portion of the nucleotide sequence encoding anRBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE),RBM15_(L) or MKL1 protein. A fragment of an RBM15-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L) or MKL1 nucleotidesequence may encode a biologically active portion of an RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L) orMKL1 protein, or it may be a fragment that can be used as ahybridization probe or PCR primer using methods disclosed below. Abiologically active portion of an RBM15-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L) or MKL1 proteincan be prepared by isolating a portion of one of the RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L),and MKL1 nucleotide sequences of the invention, expressing the encodedportion of the RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S),RBM15_(S+AE), RBM15_(L) or MKL1 protein (e.g., by recombinant expressionin vitro), and assessing the activity of the encoded portion of theRBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE),RBM15_(L), and MKL1 protein. Nucleic acid molecules that are fragmentsof an RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S),RBM15_(S+AE), RBM15_(L), and MKL1 nucleotide sequence comprise at leastabout 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4500,5000, 5500, 6000, or 6500 nucleotides, or up to the number ofnucleotides present in a full-length nucleotide sequence disclosedherein (for example, 6836, 923, 1034, 3312, 3423, 3383, and 4447nucleotides for SEQ ID NOS: 1, 3, 5, 7, 9, 11, and 13, respectively)depending upon the intended use.

It is understood that isolated fragments include any contiguous sequencenot disclosed prior to the invention as well as sequences that aresubstantially the same and which are not disclosed. Accordingly, if anisolated fragment is disclosed prior to the present invention, thatfragment is not intended to be encompassed by the invention. When asequence is not disclosed prior to the present invention, an isolatednucleic acid fragment is at least about 12, 15, 20, 25, or 30 contiguousnucleotides. Other regions of the nucleotide sequence may comprisefragments of various sizes, depending upon potential homology withpreviously disclosed sequences.

A fragment of an RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L) or MKL1 nucleotide sequence thatencodes a biologically active portion of an RBM15-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L), or MKL1 proteinof the invention will encode at least about 15, 25, 30, 50, 75, 100,125, 150, 175, 200, 250, or 300 contiguous amino acids, or up to thetotal number of amino acids present in a full-length RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L),or MKL1 protein of the invention (for example, 1883, 17, 25, 969, 977,957, and 931 amino acids for SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 14,respectively). Fragments of an RBM15-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L) and MKL1nucleotide sequence that are useful as hybridization probes for PCRprimers generally need not encode a biologically active portion of anRBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE),RBM15_(L), and MKL1 protein, respectively.

Nucleic acid molecules that are variants of the nucleotide sequencesdisclosed herein are also encompassed by the present invention.“Variants” of the RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L), and MKL1 nucleotide sequencesinclude those sequences that encode the RBM15-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L), and MKL1proteins, respectively, disclosed herein but that differ conservativelybecause of the degeneracy of the genetic code. These naturally occurringallelic variants can be identified with the use of well-known molecularbiology techniques, such as polymerase chain reaction (PCR) andhybridization techniques as outlined below. Variant nucleotide sequencesalso include synthetically derived nucleotide sequences that have beengenerated, for example, by using site-directed mutagenesis but whichstill encode the RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L), or MKL1 protein disclosed in thepresent invention as discussed below. Generally, nucleotide sequencevariants of the invention will have at least about 45%, 55%, 65%, 75%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97, 98%, or 99% identity to aparticular nucleotide sequence disclosed herein. A variant RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L) orMKL1 nucleotide sequence will encode a variant RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L),or MKL1 protein, respectively, that has an amino acid sequence having atleast about 45%, 55%, 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97, 98%, or 99% identity to the amino acid sequence of an RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L),or MKL1 protein disclosed herein.

In addition to the RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L) and MKL1 nucleotide sequences shownin SEQ ID NOS: 1, 3, 5, 7, 9, 11, and 13 , it will be appreciated bythose skilled in the art that DNA sequence polymorphisms that lead tochanges in the amino acid sequences of RBM5-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L), or MKL1 proteinsmay exist within a population (e.g., the human population). Such geneticpolymorphism in an RBM15-MKL1, MKL1-RBM15, RBM15, and MKL1 gene mayexist among individuals within a population due to natural allelicvariation. An allele is one of a group of genes that occur alternativelyat a given genetic locus. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules comprising an openreading frame encoding an RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L), or MKL1 protein, preferably amammalian RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S),RBM¹⁵ _(S+AE), RBM15_(L), or MKL1 protein. As used herein, the phrase“allelic variant” refers to a nucleotide sequence that occurs at anRBM15-MKL1, MKL1-RBM15, RBM15, and MKL1 locus or to a polypeptideencoded by the nucleotide sequence. Such natural allelic variations cantypically result in 1-5% variance in the nucleotide sequence of theRBM15-MKL1, MKL1-RBM15, RBM15, and MKL1 gene. Any and all suchnucleotide variations and resulting amino acid polymorphisms orvariations in an RBM15-MKL1, MKL1-RBM15, RBM15, and MKL1 amino acidsequence that are the result of natural allelic variation and that donot alter the functional activity of RBM15-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L), and MKL1 proteinsare intended to be within the scope of the invention.

Moreover, nucleic acid molecules encoding RBM15-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L), and MKL1 proteinsfrom other species (RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L), and MKL1 homologues), which have anucleotide sequence differing from that of the RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L),and MKL1 sequences disclosed herein, are intended to be within the scopeof the invention. For example, nucleic acid molecules corresponding tonatural allelic variants and homologues of the human RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L)and MKL1 cDNAs of the invention can be isolated based on their identityto the human RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S),RBM15_(S+AE), RBM15_(L), and MKL1 nucleic acids disclosed herein usingthe human cDNA, or a portion thereof, as a hybridization probe accordingto standard hybridization techniques under stringent hybridizationconditions as disclosed herein.

In addition to naturally occurring allelic variants of the RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L),and MKL1 sequences that may exist in the population, the skilled artisanwill further appreciate that changes can be introduced by mutation intothe nucleotide sequences of the invention thereby leading to changes inthe amino acid sequence of the encoded RBM15-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L), and MKL1proteins, respectively, without altering the biological activity of theRBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE),RBM15_(L), and MKL1 proteins. Thus, an isolated nucleic acid moleculeencoding an RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S),RBM15_(S+AE), RBM15_(L), or MKL1 protein having a sequence that differsfrom that of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14, respectively, can becreated by introducing one or more nucleotide substitutions, additions,or deletions into the corresponding nucleotide sequence disclosedherein, such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Such variant nucleotide sequences are alsoencompassed by the present invention.

For example, preferably, conservative amino acid substitutions may bemade at one or more predicted, preferably nonessential amino acidresidues. A “nonessential” amino acid residue is a residue that can bealtered from the wild-type sequence of an RBM15-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L), or MKL1 protein(e.g., the sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14,respectively) without altering the biological activity, whereas an“essential” amino acid residue is required for biological activity. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Suchsubstitutions would not be made for conserved amino acid residues, orfor amino acid residues residing within a conserved motif.

Alternatively, variant RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L), and MKL1 nucleotide sequences can bemade by introducing mutations randomly along all or part of anRBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE),RBM15_(L) or MKL1 coding sequence, such as by saturation mutagenesis,and the resultant mutants can be screened for RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L),or MKL1 biological activity to identify mutants that retain activity.Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

Thus, the nucleotide sequences of the invention include the sequencesdisclosed herein as well as fragments and variants thereof. TheRBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE),RBM15_(L), and MKL1 nucleotide sequences of the invention, and fragmentsand variants thereof, can be used as probes and/or primers to identifyand/or clone RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S),RBM15_(S+AE), RBM15_(L), and MKL1 homologues in other cell types, e.g.,from other tissues, as well as homologues from other mammals. Suchprobes can be used to detect transcripts or genomic sequences encodingthe same or identical proteins.

In this manner, methods such as PCR, hybridization, and the like can beused to identify such sequences having substantial identity to thesequences of the invention. See, for example, Sambrook et al. (1989)Molecular Cloning: Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NY).RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE),RBM15_(L), and MKL1 nucleotide sequences isolated based on theirsequence identity to the RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L) and MKL1 nucleotide sequences setforth herein or to fragments and variants thereof are encompassed by thepresent invention.

In a hybridization method, all or part of a known RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L) orMKL1 nucleotide sequence can be used to screen cDNA or genomiclibraries. Methods for construction of such cDNA and genomic librariesare generally known in the art and are disclosed in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.). The so-called hybridizationprobes may be genomic DNA fragments, cDNA fragments, RNA fragments, orother oligonucleotides, and may be labeled with a detectable group suchas ³²P, or any other detectable marker, such as other radioisotopes, afluorescent compound, an enzyme, or an enzyme co-factor. Probes forhybridization can be made by labeling synthetic oligonucleotides basedon the known RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S),RBM15_(S+AE), RBM15_(L), and MKL1 nucleotide sequence disclosed herein.Degenerate primers designed on the basis of conserved nucleotides oramino acid residues in a known RBM15-MKL1, MKL1-RBM15_(S),MKL1-RBM15_(S+AE), RBM₁₅ _(S), RBM15_(S+AE), RBM15_(L) and MKL1nucleotide sequence or encoded amino acid sequence can additionally beused. The probe typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, preferablyabout 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250,300, 350, or 400 consecutive nucleotides of an RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L),and MKL1 nucleotide sequence of the invention or a fragment or variantthereof. Preparation of probes for hybridization is generally known inthe art and is disclosed in Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), herein incorporated by reference.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences having at least about 60%, 65%, 70%,preferably 75% identity to each other typically remain hybridized toeach other. Such stringent conditions are known to those skilled in theart and can be found in Current Protocols in Molecular Biology (JohnWiley & Sons, New York (1989)), 6.3.1-6.3.6. A preferred, non-limitingexample of stringent hybridization conditions is hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 50-65° C. In another preferredembodiment, stringent conditions comprise hybridization in 6×SSC at 42°C., followed by washing with 1×SSC at 55° C. Preferably, an isolatednucleic acid molecule that hybridizes under stringent conditions to anRBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE),RBM15_(L), or MKL1 nucleotide sequence of the invention corresponds to anaturally occurring nucleic acid molecule. As used herein, a “naturallyoccurring” nucleic acid molecule refers to an RNA or DNA molecule havinga nucleotide sequence that occurs in nature (e.g., encodes a naturalprotein).

Thus, in addition to the RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L), and MKL1 nucleotide sequencesdisclosed herein and fragments and variants thereof, the isolatednucleic acid molecules of the invention also encompass homologous DNAsequences identified and isolated from other cells and/or organisms byhybridization with entire or partial sequences obtained from theRBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE),RBM15_(L), and MKL1. By inserting any of the nucleotide sequences of thepresent invention into an appropriate vector, one skilled in the art canreadily produce large quantities of the specific sequence.Alternatively, the RBM15-MKL1, MKL1-RBM15_(S) and MKL1-RBM15_(S+AE),nucleotide sequences of the invention can be further utilized in methodsof producing the RBM15-MKL1, MKL1-RBM15_(S) and MKL1-RBM15_(S+AE) fusionproteins, respectively, by introduction of the appropriate codingsequence into a host/vector expression system. There are numeroushost/vectors systems available for the propagation of nucleotidesequences and/or the production of expressed proteins. These include,but are not limited to, plasmid and viral vectors, and prokaryotic andeukaryotic host. One skilled in the art can readily adapt anyhost/vector system which is capable of propagating or expressingheterologous DNA to produce or express the sequences of the presentinvention. Of course, the RBM15-MKL1, MKL1-RBM15_(S) andMKL1-RBM15_(S+AE) fusion proteins or polypeptides derived therefrom mayalso be produced by other means known in the art such as, for example,chemical synthesis or in vitro transcription/translation.

Also provided by the present invention are an isolated RBM15-MKL1 fusionprotein (SEQ ID NO: 2), an isolated MKL1-RBM15_(S) fusion protein (SEQID NO: 4), and an isolated MKL1-RBM15_(S+AE) fusion protein (SEQ ID NO:6), an isolated RBM15_(S) protein (SEQ ID NO: 8), an isolatedRBM15_(S+AE) protein (SEQ ID NO: 10), an isolated RBM15_(L) protein (SEQID NO: 12), and an isolated MKL1 protein (SEQ ID NO: 14), which areencoded by their cognate nucleotides, that is, by SEQ ID NO: 1, 3, 5, 7,9, 11, and 13, respectively. Synthetic oligopeptides derived from SEQ IDNO: 2, 4, 6, 8, 10, 12, and 14 are also provided in this embodiment ofthe invention.

An “isolated” or “purified” nucleic acid molecule or protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For purposes of the invention,“isolated” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, the isolatednucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived.

An isolated protein that is substantially free of cellular materialincludes preparations of RBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE),RBM15_(S), RBM15_(S+AE), RBM15_(L), or MKL1 protein having less thanabout 30%, 20%, 10%, or 5% (by dry weight) of non-like protein (alsoreferred to herein as a “contaminating protein”). When the RBM15-MKL1,MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE), RBM15_(L),or MKL1 protein or biologically active portion thereof is recombinantlyproduced, preferably, culture medium represents less than about 30%,20%, 10%, or 5% of the volume of the protein preparation. WhenRBM15-MKL1, MKL1-RBM15_(S), MKL1-RBM15_(S+AE), RBM15_(S), RBM15_(S+AE),RBM15_(L), or MKL1 protein is produced by chemical synthesis, preferablythe protein preparations have less than about 30%, 20%, 10%, or 5% (bydry weight) of chemical precursors or non-like chemicals.

In Example 1, the present invention provides evidence that thenucleotide sequences containing the RBM15-MKL1 and MKL1-RBM15 fusiongenes are present in patients with t(1;22) AMKL. Based on thisobservation, the present invention provides methods of assaying for thepresence of nucleotide sequences containing the RBM15-MKL1 andMKL1-RBM15 fusions in a sample and thus provides an assay for thedetection of t(1;22) leukemias, as explained in Example 1. The methodsof the invention can involve any means known in the art for detectingthe presence of specific nucleotide sequences in a sample including, butnot limited to, nucleic acid hybridization and detection methods (e.g.,“Southerns,” “Northerns” and the like) fluorescence in situhybridization (FISH) and detection methods, or polymerase chain reaction(PCR) amplification and detection methods, particularly reversetranscriptase-polymerase chain reaction amplification (RT-PCR).

One example of the assay methods of the present invention which are usedto detect RBM15-MKL1 or MKL1-RBM15 fusion gene are based on thepreferential amplification of sequences within a sample which containthe nucleotide sequences encoding the RBM15-MKL1, MKL1-RBM15_(S) orMKL1-RBM15_(S+AE) fusion proteins. In one embodiment of the invention,RT-PCR is utilized to detect the t(1,22) rearrangement that isassociated with AMLK. The method involves the use of RT-PCR to detectthe presence of transcripts from the RBM15-MKL1 or MKL1-RBM15 fusiongenes. The method involves reverse transcription via reversetranscriptase of an RNA sample from a patient to produce cDNA. Forreverse transcription, an oligo-dT primer can be use, or alternatively,a primer designed to specifically anneal to RBM15-MKL1 mRNA,MKL1-RBM15_(S)mRNA, or MKL1-RBM15_(S+AE)mRNA can be employed to primecDNA synthesis. Such primers can be designed from the nucleotidesequences of the invention as set forth in SEQ ID NOS: 1, 3, and 5 usingmethods known to those of ordinary skill in the art. Then, PCRamplification of the cDNA can be performed utilizing primers designed toamplify at least a portion of the nucleotide sequences of to RBM15-MKL1,MKL1-RBM15_(S) or MKL1-RBM15_(S+AE). The amplified cDNA can be detectedby methods known in the art such as, for example, agarose gelelectrophoresis and ethidium bromide staining. The detection of thedesired cDNA corresponding to at least a portion of the RBM15-MKL1,MKL1-RBM15_(S) or MKL1-RBM15_(S+AE) indicates that the sample is from apatient with the t(1,22) rearrangement.

The methods of the invention involve the use of PCR amplification,particularly RT-PCR. Methods for PCR amplification are known in the art.Oligonucleotide primers can be designed for use in PCR reactions toamplify corresponding DNA sequences from genomic DNA or cDNA extractedfrom any organism of interest. Methods for PCR amplification and fordesigning PCR primers are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See alsoInnis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Other knownmethods of PCR that can be used in the methods of the invention include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,mixed DNA/RNA primers, vector-specific primers, partially mismatchedprimers, and the like.

In addition to methods which rely on the amplification of a targetsequence, the present invention further provides methods for identifyingnucleic acids containing the RBM15-MKL1 fusion gene which do not requiresequence amplification and are based on the known methods of Southern(DNA:DNA) and Northern (DNA:RNA) blot hybridizations, and FISH ofchromosomal material, using probes derived from the nucleotide sequencesof the invention. Additionally, other nucleotide sequences ofchromosomes 1 and 22 that are known to art and for which are disclosedherein to occur in the vicinity of the chromosomal breakpoint for thet(1;22)(p13;q13) chromosomal translocation event associated with AMKLcan be used in the methods of the invention. That is, nucleic acidprobes can be used that comprise nucleotide sequences in proximity tothe t(1;22)(p13;q13) chromosomal translocation event, or breakpoint. By“in proximity to” is intended within about 10 kilobases (kb) of thet(1;22) breakpoint. Such other nucleotide sequences include, but are notlimited to, RP11-260A24 (Accession no. AC025987), RP5-1042K10 (Accessionno. AL022238), RP11-313L7, RP5-1125M8 (Accession no. AL356387),RP4-665N5, RP4-743K1, RP11-50F6, RP3-377F16 (Accession no. Z93783),RP4-591N18 (Accession no. AL031594), RP1-229A8 (Accession no. Z86090),and RP4-735G18 (Accession no. AL096703). The clones not identified withAccession numbers are also available from the Roswell Park CancerInstitute (RPCI-BAC library).

In another embodiment of the invention, methods are provided detectingthe t(1,22) rearrangement involving FISH (fluorescence in situhybridization ) of human chromosomal material. For example, a probe thatis comprised of nucleotide sequences that span the breakpoint in eithera wild-type chromosome 1 or 22 can be used. Such a probe can hybridizeto both derivative chromosomes in the case of a t(1,22) rearrangement.Alternatively, two probes, each labeled with a different detectionreagent, can be utilized. The first probe is capable of hybridizing tosequences within the region of chromosomal band 1p13, and the secondprobe is capable of binding to the region of chromosomal band 22q13. Thetwo probes are also selected such that, in a t(1,22) rearrangement, bothprobes hybridize to the same derivative chromosome, whether it bechromosome 1 or 22. In such a case, a signal from each of the probes isobserved on the same chromosome.

The nucleic acid probes of the present invention include DNA as well asRNA probes, such probes being generated using techniques known in theart (Sambrook et al., eds., Molecular Cloning, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1989)). A skilled artisan can employ suchknown techniques using the RBM15_(S), RBM15_(S+AE), RBM15_(L), MKL1,RBM15-MKL1, MKL1-RBM15_(S), and MKL1-RBM15_(S+AE) nucleotide sequencesherein described, or fragments thereof, as probes.

For nucleic acid probes, examples of detection reagents include, but arenot limited to radiolabeled probes, enzymatic labeled probes (horseradish peroxidase, alkaline phosphatase), and affinity labeled probes(biotin, avidin, or steptavidin). For antibodies, examples of detectionreagents include, but are not limited to, labeled secondary antibodies,or in the alternative, if the primary antibody is labeled, thechromophoric, enzymatic, or antibody binding reagents which are capableof reacting with the labeled antibody. One skilled in the art willreadily recognize that the antibodies and nucleic acid probes describedin the present invention can readily be incorporated into one of theestablished kit formats which are well known in the art.

The samples used in the detection methods of the present inventioninclude, but are not limited to, cells or tissues, protein, membrane, ornucleic acid extracts of the cells or tissues, and biological fluidssuch as blood, serum, and plasma. The sample used in the methods of theinvention will vary based on the assay format, nature of the detectionmethod, and the tissues, cells or extracts which are used as the sample.Methods for preparing protein extracts, membrane extracts or nucleicacid extracts of cells are well known in the art and can be readily beadapted in order to obtain a sample which is compatible with the methodutilized (see, for example, K. Budelier et al., Chapter 2, “Preparationand Analysis of DNA,” M. E. Greenberg et al., Chapter 4, “Preparationand Analysis of RNA” and M. Moos et al., Chapter 10, “Analysis ofProteins,” in Ausubel et al., Current Protocols in Molecular Biology,Wiley Press, Boston, Mass. (1993)). One preferred type of sample whichcan be utilized in the present invention is derived from isolatedlymphoma cells. Such cells can be used to prepare a suitable extract orcan be used in procedures based on in situ analysis.

The present invention further provides antibodies specific to epitopesof the RBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE) fusion proteinsand methods of detecting the RBM15-MKL1, MKL1-RBM15_(S), orMKL1-RBM15_(S+AE) fusion proteins, or any combination thereof, that relyon the ability of these antibodies to selectively bind to specificportions of the RBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE)proteins that are unique to that fusion protein. Such antibodies do notbind preferentially to the RBM15 or MKL1 proteins.

The present invention further provides methods of detecting the presenceof at least one of the RBM15-MKL1, MKL1-RBM15_(S), and MKL1-RBM15_(S+AE)fusion proteins. Antibodies can be prepared which recognize a fusionprotein of the invention. Such antibodies can be used to detect thepresence of the fusion protein in samples from human cells. The methodsof the invention involve the use of antibodies that bind to at least oneof the fusion proteins of the invention and antibody detection systemsthat are known to those of ordinary skill in the art. Such methods finduse in diagnosis and treatment of AMKL, for example, to determine ifparticular cells or tissues express the RBM15-MKL1, MKL1-RBM15_(S),and/or the MKL1-RBM15_(S+AE) fusion proteins.

Conditions for incubating an antibody with a test sample vary dependingon the format employed for the assay, the detection methods employed,the nature of the test sample, and the type and nature of the antibodyused in the assay. One skilled in the art will recognize that any one ofthe commonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedouchterlony, or rocket inmunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention. Examples of suchassays can be found in Chard, T., An Introduction to Radioimmunoassayand Related Techniques, Elsevier Science Publishers, Amsterdam, TheNetherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

In another embodiment of the immunoassays of the invention, theanti-RBM15-MKL1 antibody, the anti-MKL1-RBM15_(S) antibody, or theanti-MKL1-RBM15_(S+AE) antibody is immobilized on a solid support.Examples of such solid supports include, but are not limited to,plastics such as polycarbonate, complex carbohydrates such as agaroseand sepharose, and acrylic resins, such as polyacrylamide and latexbeads. Techniques for coupling antibodies to such solid supports arewell known in the art (see, for example, Weir, D. M. et al., Handbook ofExperimental Immunology, 4th Ed., Blackwell Scientific Publications,Oxford, England, Chapter 10 (1986)).

Additionally, one or more of the antibodies used in the above describedmethods can be detectably labeled prior to use. Antibodies can bedetectably labeled through the use of radioisotopes, affinity labels(such as biotin, avidin, etc.), enzymatic labels (such as horse radishperoxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITCor rhodamine, etc.), paramagnetic atoms, etc. Procedures foraccomplishing such labeling are well-known in the art; see, for example,Stemberger, L. A. et al., J. Histochem. Cytochem. 18:315-333 (1970);Bayer, E. A. et al., Meth. Enzym. 62:308-315 (1979); Engrall, E. et al.,J. Immunol. 109:129-135 (1972); Goding, J. W., J. Immunol. Meth.13:215-226 (1976).

The present invention further includes methods for selectively killingcells expressing the RBM15-MKL1 fusion protein, the MKL1-RBM15_(S)fusion protein, and/or the MKL1-RBM15_(S+AE) fusion protein by, forexample, contacting a cell expressing the RBM15-MKL1, MKL1-RBM15_(S),and/or MKL1-RBM15_(S+AE) fusion protein with a toxin derivatizedantibody, wherein the antibody is capable of selectively binding to thefusion protein with only weak or no binding to non-fusion RBM15 or MKL1protein. An example of such an antibody is toxin derivatized antibodieswhich bind to the RBM15-MKL1 fusion protein junction. As used herein, anantibody is said to be “toxin-derivatized” when the antibody iscovalently attached to a toxin moiety. Procedures for coupling suchmoieties to a molecule are well known in the art. The binding of a toxinderivatized antibody to a cell brings the toxin moiety into closeproximity to the cell and thereby promotes cell death. By providing suchan antibody molecule to a mammal, the cell expressing the fusion proteincan be preferentially killed. Any suitable toxin moiety may be employed;however, it is preferable to employ toxins such as, for example, thericin toxin, the cholera toxin, the diphtheria toxin, radioisotopictoxins, or membrane-channel-forming toxins.

The antibodies or toxin-derivatized antibodies of the present inventionmay be administered to a mammal intravenously, intramuscularly,subcutaneously, enterally, topically or parenterally. When administeringantibodies or peptides by injection, the administration may be bycontinuous injections, or by single or multiple injections.

The antibodies or toxin-derivatized antibodies of the present inventionare intended to be provided to recipient mammal in a “pharmaceuticallyacceptable form” in an amount sufficient to “therapeutically effective.”An amount is said to be therapeutically effective if the dosage, routeof administration, etc. of the agent are sufficient to preferentiallykill a portion of the cells expressing the RBM15-MKL1 or MKL1-RBM15fusion protein. An antibody is said to be in a “pharmacologicallyacceptable form” if its administration can be tolerated by a recipientpatient. The antibodies of the present invention can be formulatedaccording to known methods of preparing pharmaceutically usefulcompositions, whereby these materials, or their functional derivatives,are combined with a pharmaceutically acceptable carrier vehicle.Suitable vehicles and their formulation, inclusive of other humanproteins, e.g., human serum albumin, are described, for example, inRemington's Pharmaceutical Sciences, 16th ed., Osol, A., ed., Mack,Easton Pa. (1980). In order to form a pharmaceutically acceptablecomposition which is suitable for effective administration, suchcompositions will contain an effective amount of an antibody of thepresent invention together with a suitable amount of carrier. Inaddition to carriers, the antibodies of the present invention may besupplied in humanized form. Humanized antibodies may be produced, forexample by replacing an immunogenic portion of an antibody with acorresponding, but non-immunogenic portion (i.e., chimeric antibodies)(Robinson, R. R. et al., International Patent PublicationPCT/US86/02269; Akira, K. et al., European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison, S. L. etal., European Patent Application 173,494; Neuberger, M. S. et al., PCTApplication WO 86/01533; Cabilly, S. et al., European Patent Application125,023; Better, M. et al., Science 240:1041-1043 (1988); Liu, A. Y. etal., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, A. Y. et al.,J. Immunol. 139:3521-3526 (1987); Sun, L. K. et al., Proc. Natl. Acad.Sci. USA 84:214-218 (1987); Nishimura, Y. et al., Cancer Res.47:999-1005 (1987); Wood, C. R. et al., Nature 314:446-449 (1985)); Shawel al., J. Natl. Cancer Inst. 80:1553-1559 (1988).

In providing a patient with an antibody or toxin-derivatized antibody,the dosage of administered agent will vary depending upon such factorsas the patient's age, weight, height, sex, general medical condition,previous medical history, etc. In general, it is desirable to providethe recipient with a dosage of the antibody which is in the range offrom about 1 pg/kg to 10 mg/kg (body weight of patient), although alower or higher dosage may be administered.

The present invention also encompasses antisense nucleic acid molecules,i.e., molecules that are complementary to a sense nucleic acid encodinga protein, e.g., complementary to the coding strand of a double-strandedcDNA molecule, or complementary to an mRNA sequence. Accordingly, anantisense nucleic acid can hydrogen bond to a sense nucleic acid. Theantisense nucleic acid can be complementary to an entire coding strand,or to only a portion thereof, e.g., all or part of the protein codingregion (or open reading frame). An antisense nucleic acid molecule canbe antisense to a noncoding region of the coding strand of a nucleotidesequence encoding a protein of interest. The noncoding regions are the5′ and 3′ sequences that flank the coding region and are not translatedinto amino acids.

Given the coding-strand sequence encoding, for example, an RBM15-MKL1fusion protein disclosed herein (e.g., SEQ ID NO: 1), antisense nucleicacids of the invention can be designed according to the rules of Watsonand Crick base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of RBM15-MKL1 mRNA, but morepreferably is an oligonucleotide that is antisense to only a portion ofthe coding or noncoding region of RBM15-MKL1 mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of RBM15-MKL1 mRNA. A preferred antisenseoligonucleotide for selective hybridisation to fusion transcripts willinclude the region spanning the RBM15 portion of the fusion transcriptand the MKL1 portion of the fusion transcript. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45, or 50 nucleotides in length. An antisense nucleic acid of theinvention can be constructed using chemical synthesis and enzymaticligation procedures known in the art. Similarly, antisense nucleotidemolecules can be prepared for the nucleotide sequences encoding theMKL1-RBM15_(S), and MKL1-RBM15_(S+AE) fusion proteins (SEQ ID NOS: 3 and5, respectively).

For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, including, but not limited to, for example e.g., phosphorothioatederivatives and acridine substituted nucleotides. Alternatively, theantisense nucleic acid can be produced biologically using an expressionvector into which a nucleic acid has been subcloned in an antisenseorientation (i.e., RNA transcribed from the inserted nucleic acid willbe of an antisense orientation to a target nucleic acid of interest,described further in the following subsection).

When used therapeutically, the antisense nucleic acid molecules of theinvention are typically administered to a subject or generated in situsuch that they hybridize with or bind to cellular mRNA and/or genomicDNA encoding a protein of the invention to thereby inhibit expression ofthe protein, e.g., by inhibiting transcription and/or translation. Anexample of a route of administration of antisense nucleic acid moleculesof the invention includes direct injection at a tissue site.Alternatively, antisense nucleic acid molecules can be modified totarget selected cells and then administered systemically. For example,antisense molecules can be linked to peptides or antibodies to form acomplex that specifically binds to receptors or antigens expressed on aselected cell surface. The antisense nucleic acid molecules can also bedelivered to cells using the vectors described herein. To achievesufficient intracellular concentrations of the antisense molecules,vector constructs in which the antisense nucleic acid molecule is placedunder the control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

In another embodiment of the present invention, methods are provided formodulating the translation of at least one RNA selected from the groupconsisting of those RNAs encoding the RBM15-MKL1, MKL1-RBM15_(S), orMKL1-RBM15_(S+AE) fusion protein in the cell. Specifically, such methodscomprise introducing into a cell a DNA sequence which is capable oftranscribing RNA which is complimentary to the mRNA encoding either theRBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE) fusion protein. Byintroducing such a sequence into a cell, antisense RNA will be producedthat will hybridize to RBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE)mRNA and block the translation of the RBM15-MKL1 or MKL1-RBM15 fusionprotein, respectively. Antisense cloning has been described elsewhere inmore detail by Methis et al., Blood 82:1395-1401 (1993); Stein et al.,Science 261:1004-1012 (1993); Mirabella et al., Anti-Cancer Drug Design6:647-661 (1991); Rosenberg et al., Nature 313:703-706 (1985); Preiss etal., Nature 313:27-32 (1985), Melton, Proc. Natl. Acad. Sci. USA82:144-148 (1985) and Kim et al., Cell 42:129-138 (1985). Transcriptionof the introduced DNA will result in multiple copies of the antisenseRNA being generated. By controlling the level of transcription ofantisense RNA, and the tissue specificity of expression via promoterselection or gene targeting of the antisense expression sequence, oneskilled in the art can regulate the level of translation of theRBM15-MKL1, MKL1-RBM15_(S), and/or MKL1-RBM15_(S+AE) fusion proteins inspecific cells within a patient. In a related method, one or moresynthetic antisense oligonucleotides that are complementary to theRBM15-MKL1, MKL1-RBM15_(S), and/or MKL1-RBM15_(S+AE) coding sequences ofthe invention, optionally including chemical modifications designed tostabilize the oligonucleotide or enhance its uptake into cells, areadministered to cells of a patient by known methods (see, for example,R. W. Wagner, Nature 372:333-335 (1994); J. Lisziewicz et al., Proc.Natl. Acad. Sci. (USA) 90:3860-3864 (1993); S. Fitzpatrick-McElligott,Bio/Technology 10: 1036-1040 (1992); E. Uhlmann et al., Chemical Reviews90:543-583 (1990); and B. Tseng et al., Cancer Gene Therapy 1:65-71(1994)).

The invention also encompasses ribozymes, which are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. The level of expression of the RBM15-MKL1,MKL1-RBM15_(S), and/or MKL1-RBM15_(S+AE) fusion proteins can also becontrolled through the use of ribozyme technology. Ribozymes (e.g.,hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature334:585-591)) can be used to catalytically cleave RBM15-MKL1,MKL1-RBM1_(S), or MKL1-RBM15_(S+AE) mRNA transcripts to thereby inhibittranslation of RBM15-MKL1, MKL1-RBM15_(S), and MKL1-RBM15_(S+AE) mRNA,respectively. A ribozyme having specificity for an RBM15-MKL1-,MKL1-RBM15_(S)-, or MKL1-RBM₁₅ _(S+AE)-encoding nucleic acid can bedesigned based upon the nucleotide sequence of the corresponding cDNAdisclosed herein (e.g., SEQ ID NOS: 1, 3, and 5, respectively). See,e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cech et al., U.S. Pat.No. 5,116,742. Alternatively, RBM15-MKL1, MKL1-RBM15_(S), orMKL1-RBM15_(S+AE) mRNA can be used to select a catalytic RNA having aspecific ribonuclease activity from a pool of RNA molecules. See, e.g.,Bartel and Szostak (1993) Science 261:1411-1418.

The present invention further provides methods of generating transgenicanimals and transformed cell lines which contain the RBM15-MKL1,MKL1-RBM15_(S), and/or MKL1-RBM15_(S+AE) nucleotide sequences. Suchanimals and cell lines are useful as animal models for human t(1;22)leukemias. In general, methods of generating transgenic animals andtransformed cell lines are well known in the art (for example, seeGrosveld et al., Transgenic Animals, Academic Press Ltd., San Diego,Calif. (1992)). Using the nucleotide sequences disclosed herein for theRBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE) or coding sequences forthe RBM15-MKL1, MKL1-RBM15_(S), and/or MKL1-RBM15_(S+AE) fusionproteins, a skilled artisan can readily generate a transgenic animal andtransformed cell lines which contains and expresses the RBM15-MKL1fusion protein, the MKL1-RBM15_(S) fusion protein, and/orMKL1-RBM15_(S+AE) fusion protein. Transgenic animals (such as mice andpigs) which express the RBM15-MKL1 fusion gene can be used as an animalmodel for human t(1;22) leukemia. Transgenic animals which express theRBM15-MKL1 fusion protein, the MKL1-RBM15_(S) fusion protein, orMKL1-RBM15_(S+AE) fusion protein, or any combination thereof, are usefulfor determining, at the molecular level, the roles of the RBM15-MKL1,MKL1-RBM15_(S), and MKL1-RBM15_(S+AE) fusion proteins in the developmentof acute megakaryoblastic leukemia. Such animals serve as models for thedevelopment of alternative therapies for t(1;22) lymphoma.

Transformed eukaryotic cell lines that express on or more of the fusionof proteins of the invention can be used, for example, to screen foragents that are useful for treating AMKL. Preferably such cell lines aremammalian cell lines. More preferably, such cell lines are human celllines. Generally, desired agents are those that suppress or eliminatephenotypic changes that occur as a result of the expression of one ormore fusion proteins of the invention in the cell. Phenotypic changesthat occur as a result of the expression of one or more fusion proteinsof the invention in the cell include, for example, the presence of CD61and absence of peroxidase and esterase (See, e.g. Bennett et al., Ann.Intern Med 103:460-462 (1985); Skinnider L. F. et al., ActaHaematologica 98 (1): 26 (1997); Avanzi et al., J. Cell Physiol. 145:458-464 (1990); Avanzi et al., Brit. J Haematol. 69: 359 (1988)).

Such desired agents may be further screened for selectivity bydetermining whether they suppress or eliminate phenotyic changes oractivities associated with expression of unfused RBM15 and/or MKL1proteins in cells that either express such unfused proteins naturally orare engineered to express such proteins. Selective agents are thosewhich suppress or eliminate phenotypes associated with expression of thefusion protein but which do not suppress or eliminate the phenotypesassociated with the unfused RBM15 and MKL1 proteins. Typically, theagents are screened by administering the agent to the cell. It isrecognized that it is preferable that a range of dosages of a particularagent be administered to the cells to determine if the agent is usefulfor treating AMKL. Appropriate cell lines that can be used in thismethod include, but are not limited to, DAMI, MEG-01, M-07e, CMK,CHRF-288-11 and UT7 cells. In another embodiment of the presentinvention, methods are provided for identifying agents which are capableof binding to the RBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE)fusion proteins herein described. Such methods comprise (a) contacting acandidate agent with RBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE)fusion protein, or fragment thereof, and (b) determining whether thecandidate agent binds to the fusion protein. Using this method, agentswhich can be used to modulate the activity of the RBM15-MKL1,MKL1-RBM15_(S), or MKL1-RBM15_(S+AE) fusion protein can be identified.Such methods can additionally comprise an additional step to select fromthe identified agents those which do not bind RBM15 or MKL1 proteins.Such an additional step involves contacting the agent with an RBM15 orMKL1 protein, or fragment thereof and determining whether the agentbinds to the protein or fragment.

There are numerous variations of the above assays which can be used by askilled artisan without the need for undue experimentation in order toisolate agonists, antagonists, and ligands of the RBM15-MKL1,MKL1-RBM15_(S), and/or MKL1-RBM15_(S+AE) fusion protein; see, forexample, Burch, R. M., in Medications Development. Drug Discovery,Databases, and Computer-Aided Drug Design, NIDA Research Monograph 134,NIH Publication No. 93-3638, Rapaka, R. S., and Hawks, R. L., eds., U.S.Dept. of Health and Human Services, Rockville, Md. (1993), pages 37-45.For example, an idiotypic antibody to RBM15-MKL1, MKL1-RBM15_(S), orMKL1-RBM15_(S+AE) fusion protein can be used to co-precipitate fusionprotein-bound agents in the purification and characterization of suchagents. Harlow, E., et al., Chapter 11 in Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring harbor, N.Y.(1988), pages 421-470. Further, an anti-idiotypic antibody toRBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE) can be used to designsynthetic RBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE) ligands.Ertl, H., et al., Vaccine 6:80-84 (1988); Wolff, M. E., in MedicationsDevelopment: Drug Discovery, Databases, and Computer-Aided Drug Design,NIDA Research Monograph 134, NIH Publication No. 93-3638, Rapaka, R. S.,and Hawks, R. L., eds., U.S. Dept. of Health and Human Services,Rockville, Md. (1993), pages 46-57. In addition, an anti-idiotypicantibody to the RBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE) fusionproteins, the RBM15-MKL1, MKL1-RBM15_(S), or MKL1-RBM15_(S+AE) fusionproteins, or a fragment thereof containing the active (ligand binding)site of the fusion protein, can be used to screen an expression libraryfor genes encoding proteins which bind the fusion protein.

Alternatively, cells expressing the RBM15-MKL1, MKL1-RBM15_(S), orMKL1-RBM15_(S+AE) fusion proteins on their surfaces can be used toscreen expression libraries or synthetic combinatorial oligopeptidelibraries. Cwirla, S. E., et al., Proc. Natl. Acad. Sci. (USA)87:6378-6382 (1990); Houghten, R. A., et al., Nature 354:84-86 (1991);Houghten, R. A., et al., in Medications Development: Drug Discovery,Databases, and Computer-Aided Drug Design, NIDA Research Monograph 134,NIH Publication No. 93-3638, Rapaka, R. S., and Hawks, R. L., eds., U.S.Dept. of Health and Human Services, Rockville, Md. (1993), pages 66-74.In particular, cells that have been genetically engineered to expressand display the RBM15-MKL1, MKL1-RBM15_(S), and/or MKL1-RBM15_(S+AE)fusion protein via the use of the nucleic sequences of the invention arepreferred in such methods, as host cell lines may be chosen which aredevoid of related receptors. Hartig, P. R., in Medications Development:Drug Discovery, Databases, and Computer-Aided Drug Design, NIDA ResearchMonograph 134, NIH Publication No. 93-3638, Rapaka, R. S., and Hawks, R.L., eds., U.S. Dept. of Health and Human Services, Rockville, Md.(1993), pages 58-65.

The agents screened in the above assay can be, but are not limited to,small molecules, peptides, carbohydrates, or vitamin derivatives. Theagents can be selected and screened at random or rationally selected ordesigned using protein modeling techniques. For random screening, agentssuch as peptides or carbohydrates are selected at random and are assayedfor their ability to bind to the pseudogene peptide. Alternatively,agents may be rationally selected or designed. As used herein, an agentis said to be “rationally selected or designed” when the agent is chosenbased on the configuration of the pseudogene peptide. For example, oneskilled in the art can readily adapt currently available procedures togenerate peptides capable of binding to a specific peptide sequence inorder to generate rationally designed antipeptide peptides, see, forexample, Hurby et al., “Application of Synthetic Peptides: AntisensePeptides,” in Synthetic Peptides: A User's Guide, W. H. Freeman, NewYork (1992), pp. 289-307; and Kaspczak et al., Biochemistry 28:9230-2938(1989).

Using the above procedures, the present invention provides agentscapable of binding to the the RBM15-MKL1, MKL1-RBM15_(S), and/orMKL1-RBM15_(S+AE) fusion proteins, produced by a method comprising thesteps of (a) contacting said agent with the the RBM15-MKL1,MKL1-RBM15_(S), and/or MKL1-RBM15_(S+AE) fusion protein, or a fragmentthereof, and (b) determining whether said agent binds to the RBM15-MKL1,MKL1-RBM15_(S), or MKL1-RBM15_(S+AE) fusion protein. Additional step(s)to determine whether such binding is selective for the fusion proteinrelative to the corresponding unfused RBM15 and MKL1 proteins may alsobe employed.

The materials used in the above assay methods (both nucleic acid andprotein based) are ideally suited for the preparation of a kit. Forexample, for amplification based detection systems, the inventionprovides a compartmentalized kit to receive in close confinement, one ormore containers which comprises (a) a first container comprising one ormore of the amplification primers of the present invention, and (b) oneor more other containers comprising one or more of the following: asample reservoir, amplification reagents, wash reagents, and detectionreagents.

For antibody based detection systems, the present invention provides acompartmentalized kit to receive in close confinement, one or morecontainers which comprises (a) a first container comprising an antibodycapable of binding to the RBM15-MKL1, MKL1-RBM15_(S), orMKL1-RBM15_(S+AE) fusion protein and (b) one or more other containerscomprising one or more of the following: wash reagents and reagentscapable of detecting the presence of bound antibodies from the first andthe second containers.

The invention further provides a kit compartmentalized to receive inclose confinement one or more containers which comprises (a) a firstcontainer comprising an antibody capable of binding to an epitope whichis present in the fusion junction of the RBM15-MKL1, MKL1-RBM15_(S), orMKL1-RBM15_(S+AE) fusion protein and which is not present in either ofthe two non-fusion proteins; and (b) one or more other containerscomprising one or more of the following: wash reagents and reagentscapable of detecting the presence of bound antibodies from the firstcontainer.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allow one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers may include a container which will accept thetest sample, a container which contains the antibodies or probes used inthe assay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or the hybridized probe. Anydetection reagents known in the art can be used including, but notlimited to those described supra.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL EXAMPLE 1 Fusion of RNA Recognition Motif-Encoding Gene,RBM15, to the SAP DNA-Binding Domain Gene, MegaKaryoblastic Leukemia-1(MKL1), in Acute Megakaryoblastic Leukemias with t(1;22)(p13;q13)Summary

Acute megakaryoblastic leukemia (AMKL) in young children is almostinvariably caused by leukemic blasts harboring t(1;22)(p13;q13)(Carroll, A. et al., Blood 78:748-752 (1991); Lion, T. et al., Blood79:3325-3330 (1992); Bernstein, J. et al., Leukemia 14:216-218 (2000)).Despite its remarkable disease specificity and a lack of knowledge ofAMKL pathogenesis (Cripe, L. D. & Hromas, R., Semin. Hematol. 35:200-209(1998)), t(1;22) has yet to be characterized molecularly. Disclosedherein is the identification of the reciprocal fusion transcriptsderived from two novel genes, RNA-binding motif protein-15 (RBM15) atchromosome 1p13 and Megakaryoblastic Leukemia-1 (MKL1) at 22q13, as theconsequence of t(1;22). RBM15, detected in three isoforms—RBM15_(L),RBM15_(S), and RBM15_(S+AE)—contains three RNA recognition motifs (RRM)(Burd, C. G. & Dreyfuss, G., Science 265:615-621 (1994)) and a Spenparalog and ortholog C-terminal (SPOC) domain (Wiellette, E. L. et al.,Development 126:5373-5385 (1999)), thus showing significant homology tospen, a homeotic Drosophila gene capable of enhancing Ras/MAP kinasesignaling (Wiellette, E. L. et al., Development 126:5373-5385 (1999);Rebay, I. et al., Genetics 154:695-712 (2000); Chen, F. & Rebay, I.,Curr. Biol. 10:943-946 (2000); Kuang, B. et al., Development127:1517-1529 (2000)). MKL1 contains a SAP (SAF-A/B, Acinus and PIAS)DNA-binding motif (Aravind, L. & Koonin, E. V., Trends Biochem. Sci.25:112-114 (2000)) that in homologous proteins such as SAF-B functionsto recruit domains involved in chromatin remodeling, transcriptionalcontrol, and pre-mRNA processing to the matrix attachment regions (MAR)of transcriptionally active chromatin, effectively couplingtranscription and splicing (Naylor, O. et al., Nucleic Acids Res.26:3542-3549 (1998)). Although both reciprocal fusion transcripts areexpressed in AMKL, RBM15-MKL1, from the der(22) chromosome, encodes allputative functional motifs of each gene and is the candidate oncogene oft(1;22), through a mechanism that may involve deregulation of RNAprocessing and/or Hox and Ras/MAP kinase signaling.

Description

Cloning of chromosomal translocations has led to identification ofpathogenically relevant oncogenic fusion transcripts and proteins inspecific subsets of acute nonlymphocytic leukemia (ANLL), such aspromyelocytic leukemia-retinoic acid receptor alpha fusion gene(PML-RARα) in acute promyelocytic leukemia (FAB-M3 subtype), acutemyeloid leukemia 1-eight twenty one fusion gene (AML1-ETO) in ANLL withmaturation (FAB-M2), and various mixed lineage leukemia (MLL) genefusions in acute myelomonocytic and monocytic leukemias (FAB-M4 and -M5)(Melnick, A. & Licht, J. D., Blood 93:3167-3215 (1999); Downing, J. R.,Br. J. Haematol. 106:296-308 (1999); Rowley, J. D., Semin. Hematol.36:59-72 (1999); Look, A. T., Science 278:1059-1064 (1997); Faretta, M.,Di Croce, L. & Pelicci, P. G., Sem. Hematol. 38:42-53 (2001)). Despitethese significant advances, little is known about the genetic mechanismsunderlying acute leukemias of the megakaryoblastic (platelet precursor)lineage (AMKL, FAB-M7) (Cripe, L. D, infra). Almost invariably, AMKL innon-Down syndrome infants and young children harbor thet(1;22)(p13;q13), in most cases as the sole cytogeneticabnormality(Carroll, A. et al; Lion, T. et al., and Bernstein, J. etal., infra). Phenotypically, AMKL presents de novo (i.e., without aso-called preleukemic stage), with a large leukemia cell mass, andfrequent fibrosis of bone marrow and other organs. Progression isusually rapid despite therapy, with a median overall patient survival ofonly 8 months.

To clone t(1;22), a fluorescence in situ hybridization (FISH)-basedpositional cloning strategy was used to define the 1p13 and 22q13breakpoints . Bacterial artificial chromosome (BAC) clones mapping toeach chromosomal band were selected using the public and Celera humansequence databases and used pairwise in a series of two-color, two-probeFISH analyses of metaphase chromosomes from t(1;22)-containing leukemiablasts to identify closely flanking clones. With this strategy, a singlechromosome 22 BAC clone, RP5-1042K10, was found that hybridized to boththe der(1) and der(22) chromosomes formed by the reciprocal balancedt(1;22) and thus contained the altered 22q13 gene locus. Additional FISHexperiments using DNA subfragments of RP5-1042K10 prepared bylong-distance PCR (LD-PCR) methods allowed refinement of the chromosome22 breakpoint to within the most telomeric gene in this clone, andrevealed that all cases with t(1;22) possessed genomic breakpoints in asingle ˜28-kb intron of this gene. The same approach demonstrated thebreakpoint on chromosome 1p13 to be encompassed by BAC clone RP11-260A24. After complete annotation of the sequence by combiningentries in the public and Celera databases, the 1p13 breakpoint wassublocalized to within an ˜6-kb genomic interval (extending fromnucleotide 1,798,871 to 1,804,858 in Celera scaffold GA_x2HTBL4WN8M)using RP11-260A24 LD-PCR subfragments in metaphase FISH. FISH analyseswith probes closely flanking the identified breakpoint regions onchromosome 1 and chromosome 22 confirmed the results of our ‘splitsignal’ analysis, highlighting both the der(1) and der(22) chromosomes.The breakpoint-spanning clones from each chromosome identified by FISHanalysis are RP11-260A24 (chromosome 1) and RP5-1042K10 (chromosome 22).An additional 22q13 BAC, clone RP11-313L7, that spanned the breakpointwas subsequently also identified, the end sequences (accession nos.AQ506839 (Sp6) and AQ537696 (T7)) of which revealed a 65,460 bp overlapwith RP5-1042K10 and a 76,650 bp overlap with an additional chromosome22 clone for this region designated RP4-591N18.

Database searches using the exon sequences flanking thebreakpoint-containing intron on chromosome 22 identified an anonymoushuman brain cDNA library clone (accession no. AB037859). This 3,907-bpcDNA encoded a 2,793-nucleotide ORF, with the putative ATG initiatorcodon in a context (ATCatgC) adequate to support translationalinitiation (Kozak, M., Mammalian Genome 7:563-574 (1996)). Because RNAblot hybridizations using this clone revealed an approximately 4.5-kbtranscript expressed ubiquitously in normal human tissues, additional 5′untranslated sequence was obtained by RACE, resulting in a complete cDNAof 4,447 bp (SEQ ID NO: 13). To denote its involvement in AMKL, thisgene was named MKL1 (Megakaryoblastic Leukemia-1, official HUGONomenclature Committee designation). Motif searches of the deduced931-amino acid (aa) MKL1 protein (predicted mass, 98.9 kDa; SEQ ID NO:14) identified a bipartite nuclear localization signal (BP-NLS)(residues 14-31 of SEQ ID NO: 14; RRSLERARTEDYLKRKIR), a single SAPDNA-binding motif (Aravind, L. & Koonin, E. V., Trends Biochem. Sci. 25,112-114 (2000)) (residues 347-381 of SEQ ID NO: 14), a coiled-coilregion (residues 521-563 of SEQ ID NO: 14) that likely mediates proteinoligomerization (Lupas, A., Trends Biochem. Sci. 21, 375-382 (1996)),and a long C-terminal proline-rich segment (residues 564-811 of SEQ IDNO: 14) similar to proline-rich regions shown to act as transcriptionalactivators (Mitchell, P. J. & Tjian, R., Science 245, 372-378 (1989)).In addition, a short glutamine-rich segment (residues 264-286 of SEQ IDNO: 14; QQQQLFLQLQILNQQQQQHHNYQ) was found that is highly reminiscent ofthe more extensive glutamine-rich regions of the MLL acuteleukemia-associated transcription factor family, as well as a number ofother proteins involved in transcriptional control (Prasad, R. et al.,Oncogene 15, 549-560 (1997)). Of note, MKL1 showed significantcross-species homology to the product of a Drosophila gene ofundetermined function, CG12188 (accession no. AAF47681), exhibiting 41%identity (57% similarity) over the initial 161 amino acids of MKL1 and63% identity (76% similarity) in the SAP domains of the two proteins.

The MKL1 SAP domain shares sequence similarities with SAP domains from(a) THO1—yeast protein Tho1p, which regulates transcriptional elongationby RNA polymerase II; (b) E1B-55 kDa, a transforming adenovirus proteinthat binds and inhibits p53, and mediates nucleocytoplasmic transport ofadenoviral and cellular mRNAs; (c) PIAS1 (protein inhibitor of activatedStat1) which binds and inhibits Stat1, coactivates transcription byvarious steroid receptors, regulates RNA helicase II function, and hasalso been reported to bind wt and mutant p53; (d) SAF-B (scaffoldattachment factor B), a RRM-containing protein that binds both RNApolymerase II and a subset of serine-/arginine-rich RNA splicingfactors; and (e) ACINUS (apoptotic chromatin condensation inducer in thenucleus) which mediates chromatin condensation during programmed celldeath (reviewed in Aravind, L. & Koonin, E. V., Trends Biochem. Sci. 25,112-114 (2000)).

With the hypothesis that t(1;22) generates an oncogenic fusion analogousto breakpoint cluster region-Abelson tyrosine kinase fusion gene(BCR-ABL) or the Mixed-lineage leukemia (MLL)fusion genes in leukemias,5′ RACE was performed with total RNA from our leukemia patient samplesusing MKL1 oligonucleotide primers to identify the 1p13 fusion partner.The obtained sequences corresponded to two anonymous, partiallyoverlapping cDNA clones (accession nos. AK025596, AK022541) fromchromosome 1 that were also contained within the approximately 6-kbgenomic interval in BAC RP11-260A24 previously demonstrated by FISH tospan the 1p13 breakpoint. Due to the presence of three RNA recognitionmotifs (RRM) encoded by these sequences, the corresponding gene wasnamed RBM15 (RNA-binding motif protein-15, official HUGO NomenclatureCommittee name). Using 5′ RACE and human placenta cDNA libraryscreening, the complete RBM15 coding sequence was obtained byidentification of an ATG initiator codon 132 nucleotides (nts) upstreamof the previously deposited sequences and preceded 30 nts by an in-frameTGA stop. Sequencing of RT-PCR products obtained with RBM15-specificprimers from normal leukocyte mRNA demonstrated three transcripts thatshare an identical 2,863-bp 5′ coding sequence, differing only in theirextreme 3′ coding portions due to alternative exon usage (FIG. 1)

RBM15 contains three RRM motifs and a BP-NLS in its C-terminus, and ishighly homologous to Drosophila gene product GH11110 (accession no. AF145664) (40% identity, 56% similarity with the RRM-containing region ofRBM15 from residues 170-529; 39% identity, 54% similarity with the RBM15C-terminus from residues 714-954; percent identity determined usingBLAST 2.1.3 (Altschul, S. F. et al., Nucleic Acids Res. 25: 3389 (1997))with default parameters selected). These regions of RBM15 and DrosophilaGH11110 (also previously referred to as D. melanogaster Short spen-likeprotein-2, DmSSLP2) (Wiellette, E. L. et al., Development 126, 5373-5385(1999)) are closely related to Drosophila spen (split ends)—an RRMprotein that modulates Hox homeotic function (e.g., cooperating withAntennapedia to suppress head-like development in the thoracic region),and regulates neuronal cell fate and axon extension by enhancing Ras/MAPkinase signaling (Wiellette, E. L. et al., Development 126, 5373-5385(1999); Rebay, I. et al., Genetics 154, 695-712 (2000); Chen, F. &Rebay, I., Curr. Biol. 10, 943-946 (2000); Kuang, B. et al., Development127, 1517-1529 (2000)). Thus, the RBM15 C-terminus also contains aso-called SPOC (Spen paralog and ortholog C-terminal) domain, a 165-aaconserved motif of undetermined function found in Spen and Spen-likeproteins (Wiellette, E. L. et al., Development 126, 5373-5385 (1999))including the mammalian spen ortholog, MINT (Msx2-interacting nucleartarget), which binds homeoprotein Msx2 (Hox 8) and coregulatesosteoblast gene expression during craniofacial development (Newberry, E.P. et al., Biochemistry 38, 10678-10690 (1999)).

In t(1;22)-positive AMKL blasts, RT-PCR demonstrated expression of bothreciprocal fusion transcripts, RBM15-MKL1 and MKL1-RBM15. RT-PCRreactions using RBM15 sense (RBM15(S)-2746F) and MKL1 antisense(MKL1-204R) primers amplify a single 268-bp RBM15-MKL1 product in allpatients (nucleotides 2866 to 3133 of SEQ ID NO: 1). Using MKL1 sense(MKL1-F) and RBM15 antisense (RBM15(S)-2930R, corresponding to sequencesof the 3′ most exon unique to RBM15_(S) and RBM15_(S+AE)) primers, tworeciprocal MKL1-RBM15 fusion transcripts are detected, one (251 bp;nucleotides 411 to 661 of SEQ ID NO: 3) containing the 3′ sequences fromRBM15_(S) and the other (362 bp; nucleotides 411 to 772 of SEQ ID NO: 5)with 3′ sequences found in RBM15_(S+AE) . No MKL1-RBM15 RT-PCR productswere obtained using MKL1-F and an antisense primer specific forRBM15_(L) (RBM15(L)-1636R) in any patients examined.

The predicted RBM15-MKL1 chimeric protein encoded on the der(22)contains all putative functional motifs of each normal protein (FIG. 1).Frequent duplication of der(1) in t(1;22)-containing blasts has led tospeculation that this abnormal chromosome likely encodes the oncogenicAMKL fusion protein(s) (Carroll, A. et al.; Lion, T. et al., Bernstein,J. et al., infra.); however, a functional role for MKL1-RBM15_(S) andMKL1-RBM15_(S+AE) in leukemogenesis is unclear given they encodepredicted products of only 17 and 25 aa, respectively.

The SAP motif mediates DNA binding of proteins to the AT-rich matrixattachment regions (MAR) associated with transcriptionally activechromatin (Aravind, L. & Koonin, E. V., Trends Biochem. Sci. 25, 112-114(2000)). SAP proteins include SAF-B (Chen, F. & Rebay, I., Curr. Biol.10, 943-946 (2000)), involved in RNA processing; Acinus (Sahara, S. etal., Nature 401, 168-173 (1999)), which induces chromatin condensation;and PIAS proteins (Valdez, B. C. et al., Biochem. Biophys. Res. Commun.234, 335-340 (1997); Chung, C. D. et al., Science 278, 1803-1805 (1997);Kotaja, N. et al., Mol. Endocrinol. 14, 1986-2000 (2000)) that bind RNAhelicase II, inhibit STAT signal transduction, and modulate steroidreceptor-dependent transcription. Thus, the SAP targets a diverse set offunctional domains to MAR sequences, coupling transcription andsplicing. In addition to modulating homeotic protein functions (and inthe case of spen, enhancing Ras/MAP kinase signals), Spen familyproteins like MINT can bind specific DNA sequences via their RRMdomains, an RRM function seen also in other transcriptional regulatorssuch as sea urchin SSAP (Stage-specific activator protein) (DeAngelo, D.J. et al., Mol. Cell. Biol. 15, 1254-1264 (1995); DeFalco, J. & Childs,G., Proc. Natl. Acad. Sci. 93, 5802-5807 (1996)) and hTAFII68, anRNA/ssDNA-binding protein homologous to pro-oncoproteins TLS/FUS and EWS(Bertolotti, A. et al., EMBO J 15, 5022-5031 (1996); Bertolotti, A. etal., Oncogene 18, 8000-8010 (1999)). In RBM15-MKL1, the MKL1 SAP motifwould be expected to relocalize the RRM domains of RBM15 aberrantly tosites of transcriptionally active chromatin, targeting genes criticalfor the normal proliferation or differentiation of megakaryoblasts.

Methods

Clinical Cases

Leukemia specimens with histopathological and immunophenotypic featurestypical of AMKL were studied from five infants and young children. Allcases contained t(1;22)(p13;q13) with the exception of patient 2, whoseblasts possessed a complex t(1;6;22)(p13;p12;q13). All five specimenswere shown to contain rearrangement of RBM15 and MKL1 by RT-PCR and/orFISH analysis.

Fluorescence In Situ Hybridization (FISH)

DNA was labeled by nick translation with digoxigenin-11-dUTP and/orbiotin-16-dUTP (Roche Molecular Biochemicals). Labeled probes werecombined with sheared human DNA and hybridized to fixed interphasenuclei and metaphase cells in 50% formamide, 10% dextran sulfate and2×SSC at 37° C. and subsequently washed in a 50% formamide, 2×SSCsolution at 37° C. Hybridization signals were detected withfluorescein-labeled anti-digoxigenin (Ventana Medical Systems) fordigoxigenin-labeled probes and Texas red-avidin for biotinylated probes.Chromosomes and nuclei were stained with 4,6-diamidino-2-phenylindole(DAPI) prior to analysis.

Rapid Amplification of cDNA Ends (RACE) and DNA Sequencing

Total RNA was extracted from t(1;22)-positive frozen AMKL specimensusing RNA STAT-60 (Tel-Test, Inc.). Approximately 0.2 μg RNA was usedfor 5′ RACE experiments that identified RBM15 as the chromosome 1p13partner gene of MKL1. Reverse transcription was performed with primerMKL1-294R (SEQ ID NO: 15). After purification and tailing of the cDNA,PCR was performed with an oligo-dT anchor primer and MKL1 reverse primerMKL1-73R (SEQ ID NO: 16), using temperature cycling conditionsrecommended by the manufacturer (Roche Molecular Biochemicals) and aThermal Cycle Model 2400 (Perkin-Elmer Cetus). Twenty microliters of PCRproduct were separated on a 2% agarose gel, and specific bands werepurified (Qiaquick gel extraction kit, Qiagen, Inc.) and sequenced usingMKL1 reverse primer MKL1-59R (SEQ ID NO: 17).

RT-PCR of RBM15-MKL1 and MKL1-RBM15 Fusion Transcripts

For RBM15-MKL1 detection, 1 μg of total RNA was reverse transcribedusing primer MKL1-294R (SEQ ID NO: 15). PCR was performed using primersRBM15(S)-2746F (SEQ ID NO: 18) and MKL1-204R (SEQ ID NO: 19) and 35cycles (94° C. for 15 s, 60° C. for 30 s, 72° C. for 30 s). Primer pairMKL1-F (SEQ ID NO: 20) and MKL1-204R, designed to amplify a portion ofthe ubiquitously expressed MKL1, were included in control experiments toverify RNA quality and RT-PCR technique. PCR products were gel purified,then cycle sequenced using primers RBM15(S)-2746F and MKL1-204R. Fordetection of MKL1-RBM15 fusion transcripts, reverse transcription wasdone using an oligo-dT primer and PCR performed with primers MKL1-F (SEQID NO: 20) and RBM15(S)-2930R (SEQ ID NO: 21) (to identifyMKL1-RBM15_(S) and MKL1-RBM15_(S+AE)) or MKL1-F (SEQ ID NO: 20) andRBM15(L)-1636R (SEQ ID NO: 22) (to detect MKL1-RBM15_(L)). Amplificationof normal RBM15 sequences for quality control was performed with primerpairs RBM15(S)-2746F (SEQ ID NO: 18) and RBM15(S)-2930R (SEQ ID NO: 21)or RBM15(L)-1636R (SEQ ID NO: 22).

Northern Blot Analysis

Normal human peripheral blood leukocyte total RNA was extracted (RNEasykit, Qiagen), then treated with RNase-free DNase for 15 m at room temp.This RNA was reverse transcribed and used as the template in PCRamplifications (35 cycles: 94° C. for 10 s, 62° C. for 10 s, 68° C. for1 m) to generate cDNA fragments corresponding to probes a-d (FIG. 1).The following PCR primer pairs were used: RBM15 probe a (433 bp),RBM15-1118F (SEQ ID NO: 23) and RBM15-1551R (SEQ ID NO: 24); RBM15 probeb (318 bp), RBM15-2831F (SEQ ID NO: 25) and RBM15-3149R (SEQ ID NO: 26);RBM15 probe c (388 bp), RBM15-1616F (SEQ ID NO: 27) and RBM15-2004R (SEQID NO: 28); MKL1 probe d (449 bp), MKL1-155F (SEQ ID NO: 29) andMKL1-294R (SEQ ID NO: 15). Multiple tissue Northern blots (Clontech)containing approximately 2 μg of poly(A)+RNA prepared from normal humantissues were hybridized at 68° C. for 2 h in ExpressHyb buffer(Clontech) using these RBM15 and MKL1 cDNA probes or a β-actin probesupplied by the manufacturer. Filters were autoradiographed at −80° C.with one intensifying screen for 3 d (probes a, c and d), 1 d (probe b)or 1 h (for β-actin).

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the following embodiments.

1. An isolated nucleotide molecule comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the fusion protein having the amino acid sequence set forth in SEQ ID NO: 2; and (b) the nucleotide sequence set forth in SEQ ID NO:
 1. 